Tumor antigen specific antibodies and tlr3 stimulation to enhance the performance of checkpoint interference therapy of cancer

ABSTRACT

The present disclosure relates to a method for inhibiting cancer tumor growth in a patient by administering to the patient a therapeutic monoclonal antibody specific for a tumor associated antigen in combination with at least one immunostimulatory compound, and at least one immune homeostatic checkpoint inhibitor. Also disclosed are uses, compositions and kits thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/501,773, filed Feb. 3, 2017, which is a U.S.National Phase application under 35 U.S.C. § 371 of PCT/CA2015/050747,filed Aug. 7, 2015, which claims benefit of U.S. Provisional ApplicationNo. 62/034,915, filed on Aug. 8, 2014, the entire disclosure contents ofthese applications are herewith incorporated by reference in theirentirety into the present application.

BACKGROUND (a) Field

The subject matter disclosed generally relates to methods for inhibitingcancer tumor growth in a patient. More specifically, the method relatesto methods comprising administering to a patient a therapeuticmonoclonal antibody specific for a tumor associated antigen incombination with at least one immunostimulatory compound, and at leastone immune homeostatic checkpoint inhibitor.

(b) Related Prior Art

Quest PharmaTech has developed a series of monoclonal antibodiesspecific to tumor antigens such as CA125, MUC1, PSA, Her2/neu and othertumor associated antigens. Quest PharmaTech is developing thesemonoclonal antibody therapies as cancer immunotherapy specificallycapable of stimulating anti-tumor immunity through altered antigenprocessing and presentation stimulated by these specific antibodies.

Demonstrations studies have been completed in animals and for severalantibodies, namely AR20.5 and B43.13, in human clinical trials. Parallelto these efforts, immunologists studying the molecular events ofadaptive immunity have defined the pathways of antigen recognition byspecific T cells using T cell receptors that recognize peptide fragmentsof antigen in the context of MHC class I and II. The dynamics of anacute response require activation of second signals in addition to Tcell receptor recognition to avoid induction of tolerance. The primaryactivating second signals are the interaction between B7.1 on antigenpresenting cells (APC) and CD28 on the T cells. These second signals areinduced in the pro-inflammatory microenvironment. Additional activatingpathways have also been defined, as well as a redundant set ofcheckpoint pathways designed to limit antigen specific activation. Thesehomeostatic checkpoint signals include the interaction of CTLA4 on Tcells with B7.1 on APC and PD-1 on T cells with B7H1 on APC.

Interference with checkpoint inhibition results in prolongation andenhancement of specific immunity. This has been applied to theimmunotherapy of multiple cancer types and as reported at the 2014meeting of the American Society of Clinical Oncology, activation ofimmunity using molecules in development, as well as commercializedmolecules in the case of one anti-CTLA-4 monoclonal antibody(ipilimumab), can result in predictable clinical responses, and durablecontrol of tumor growth and, on occasion, shrinkage and elimination ofdisease in patients with advanced solid malignancy. Responses to therapyhave been associated with the presence of mutations on common tumorantigens presumably creating Neoantigens that are more prone to immuneattacks by endogenous T cells (Snyder et al ASCO Proceedings 2014abstract 3003). The performance of immune checkpoint blockade therapies,however, remains limited, with responses seen in less than 50% ofpatents and complete responses observed in only a few percent of treatedpatients.

Therefore, there is a need for method that will improve the performanceof immune checkpoint blockade therapies.

SUMMARY

According to an embodiment, there is provided a method for inhibitingcancer tumor growth in a patient comprising administering to the patienta therapeutic monoclonal antibody specific for a tumor associatedantigen in combination with at least one immunostimulatory compound, andat least one immune homeostatic checkpoint inhibitor.

The tumor-associated target antigen may be expressed on the surface of acell of the tumor.

The tumor-associated target antigen may be a soluble antigen.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an IgG antibody, an IgE antibody, or a combinationthereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to MUC1.

The antibody specific to MUC1 binds an epitope of MUC1 selected from SEQID NO: 5.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 1 and wherein a lightchain variable region of the antibody specific to MUC1 may be encoded bya nucleic acid comprising SEQ ID NO: 2.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 3 and a light chainvariable region of the antibody specific to MUC1 may be encoded by anucleic acid comprising SEQ ID NO: 4.

The antibody specific to MUC1 may be mAb-AR20.5, mAb 3C6.hIgE, mAb4H5.hIgE or a combination thereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to CA125.

The antibody specific to CA125 may be mAb-B43.13.

The immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

The TLR3 agonist may be polyIC, polyICLC (Hiltonol®).

The immune homeostatic checkpoint inhibitor an anti-PD-1 antibody, ananti-PDL-1, an anti-CTLA-4 antibody, or molecular inhibitors of thesereceptors.

The anti-PD-1 antibody may be selected from the group consisting ofnivolumab antibody, pembrolizumab antibody, pidilizumab antibody orcombinations thereof.

The anti-PDL-1 antibody may be selected from the group consisting ofB7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody,MEDI-4736 antibody, MSB0010718C antibody or combinations thereof.

The anti-CTLA-4 antibody may be selected from the group consisting ofipilimumab or tremelimumab or combinations thereof.

The therapeutic tumor associated antigen specific antibody may be murinemonoclonal antibody (xenotypic), a chimeric monoclonal antibody, ahumanized monoclonal antibody or a fully human monoclonal antibody.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have a constant region that may be of human origin.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have variable regions that are of human origin, non-humanorigin or any combination thereof.

The cancer may be selected from pancreatic cancer, breast cancer,colorectal cancer, ovarian cancer, renal cancer, prostate cancer,bladder cancer, gastrointestinal cancer, lung cancer and multiplemyeloma.

The tumor associated antigen may be CA125, folate binding protein (FBP),HER2/neu, MUC1 or PSA.

The method mat comprise the steps of:

-   -   a) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen;    -   b) administering a therapeutically effective amount of the        immunostimulatory compound after step a); and    -   c) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor, after step b).

The step b) may be performed 1 or more days after step a).

The step c) may be performed 1 or more days after step b).

According to another embodiment, there is provided a use of atherapeutic monoclonal antibody specific for a tumor associated antigenin combination with at least one immunostimulatory compound, and atleast one immune homeostatic checkpoint inhibitor for inhibiting cancertumor growth in a patient in need thereof.

The tumor-associated target antigen may be expressed on the surface of acell of the tumor.

The tumor-associated target antigen may be a soluble antigen.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an IgG antibody, an IgE antibody, or a combinationthereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to MUC1.

The antibody specific to MUC1 binds an epitope of MUC1 selected from SEQID NO: 5.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 1 and wherein a lightchain variable region of the antibody specific to MUC1 may be encoded bya nucleic acid comprising SEQ ID NO: 2.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 3 and a light chainvariable region of the antibody specific to MUC1 may be encoded by anucleic acid comprising SEQ ID NO: 4.

The antibody specific to MUC1 may be mAb-AR20.5, mAb 3C6.hIgE, mAb4H5.hIgE or a combination thereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to CA125.

The antibody specific to CA125 may be mAb-B43.13.

The immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

The TLR3 agonist may be polyIC, polyICLC (Hiltonol®).

The immune homeostatic checkpoint inhibitor an anti-PD-1 antibody, ananti-PDL-1, an anti-CTLA-4 antibody, or molecular inhibitors of thesereceptors.

The anti-PD-1 antibody may be selected from the group consisting ofnivolumab antibody, pembrolizumab antibody, pidilizumab antibody orcombinations thereof.

The anti-PDL-1 antibody may be selected from the group consisting ofB7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody,MEDI-4736 antibody, MSB0010718C antibody or combinations thereof.

The anti-CTLA-4 antibody may be selected from the group consisting ofipilimumab or tremelimumab or combinations thereof.

The therapeutic tumor associated antigen specific antibody may be murinemonoclonal antibody (xenotypic), a chimeric monoclonal antibody, ahumanized monoclonal antibody or a fully human monoclonal antibody.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have a constant region that may be of human origin.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have variable regions that are of human origin, non-humanorigin or any combination thereof.

The cancer may be selected from pancreatic cancer, breast cancer,colorectal cancer, ovarian cancer, renal cancer, prostate cancer,bladder cancer, gastrointestinal cancer, lung cancer and multiplemyeloma.

The tumor associated antigen may be CA125, folate binding protein (FBP),HER2/neu, MUC1 or PSA.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be used prior to the immunostimulatory compound.

The immunostimulatory compound may be used prior to the immunehomeostatic checkpoint inhibitor.

The monoclonal antibody specific for a tumor associated antigen may beused 1 or more days prior to the immunostimulatory compound.

The immunostimulatory compound may be used 1 or more days prior to theimmune homeostatic checkpoint inhibitor.

According to another embodiment, there is provided a composition for usein inhibiting cancer tumor growth in a patient in need thereof, thecomposition comprising a therapeutic monoclonal antibody specific for atumor associated antigen, at least one immunostimulatory compound, andat least one immune homeostatic checkpoint inhibitor.

The tumor-associated target antigen may be expressed on the surface of acell of the tumor.

The tumor-associated target antigen may be a soluble antigen.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an IgG antibody, an IgE antibody, or a combinationthereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to MUC1.

The antibody specific to MUC1 binds an epitope of MUC1 selected from SEQID NO: 5.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 1 and wherein a lightchain variable region of the antibody specific to MUC1 may be encoded bya nucleic acid comprising SEQ ID NO: 2.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 3 and a light chainvariable region of the antibody specific to MUC1 may be encoded by anucleic acid comprising SEQ ID NO: 4.

The antibody specific to MUC1 may be mAb-AR20.5, mAb 3C6.hIgE, mAb4H5.hIgE or a combination thereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to CA125.

The antibody specific to CA125 may be mAb-B43.13.

The immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

The TLR3 agonist may be polyIC, polyICLC (Hiltonol®).

The immune homeostatic checkpoint inhibitor an anti-PD-1 antibody, ananti-PDL-1, an anti-CTLA-4 antibody, or molecular inhibitors of thesereceptors.

The anti-PD-1 antibody may be selected from the group consisting ofnivolumab antibody, pembrolizumab antibody, pidilizumab antibody orcombinations thereof.

The anti-PDL-1 antibody may be selected from the group consisting ofB7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody,MEDI-4736 antibody, MSB0010718C antibody or combinations thereof.

The anti-CTLA-4 antibody may be selected from the group consisting ofipilimumab or tremelimumab or combinations thereof.

The therapeutic tumor associated antigen specific antibody may be murinemonoclonal antibody (xenotypic), a chimeric monoclonal antibody, ahumanized monoclonal antibody or a fully human monoclonal antibody.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have a constant region that may be of human origin.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have variable regions that are of human origin, non-humanorigin or any combination thereof.

The cancer may be selected from pancreatic cancer, breast cancer,colorectal cancer, ovarian cancer, renal cancer, prostate cancer,bladder cancer, gastrointestinal cancer, lung cancer and multiplemyeloma.

The tumor associated antigen may be CA125, folate binding protein (FBP),HER2/neu, MUC1 or PSA.

According to another embodiment, there is provided a kit for use ininhibiting cancer tumor growth in a patient in need thereof, the kitcomprising

-   -   a therapeutic monoclonal antibody specific for a tumor        associated antigen,    -   at least one immunostimulatory compound,    -   at least one immune homeostatic checkpoint inhibitor, and    -   instructions on how to use the kit.

The tumor-associated target antigen may be expressed on the surface of acell of the tumor.

The tumor-associated target antigen may be a soluble antigen.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an IgG antibody, an IgE antibody, or a combinationthereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to MUC1.

The antibody specific to MUC1 binds an epitope of MUC1 selected from SEQID NO: 5.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 1 and wherein a lightchain variable region of the antibody specific to MUC1 may be encoded bya nucleic acid comprising SEQ ID NO: 2.

The heavy chain variable region of the antibody specific to MUC1 may beencoded by a nucleic acid comprising SEQ ID NO: 3 and a light chainvariable region of the antibody specific to MUC1 may be encoded by anucleic acid comprising SEQ ID NO: 4.

The antibody specific to MUC1 may be mAb-AR20.5, mAb 3C6.hIgE, mAb4H5.hIgE or a combination thereof.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may be an antibody specific to CA125.

The antibody specific to CA125 may be mAb-B43.13.

The immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

The TLR3 agonist may be polyiC, polyICLC (Hiltonol®).

The immune homeostatic checkpoint inhibitor an anti-PD-1 antibody, ananti-PDL-1, an anti-CTLA-4 antibody, or molecular inhibitors of thesereceptors.

The anti-PD-1 antibody may be selected from the group consisting ofnivolumab antibody, pembrolizumab antibody, pidilizumab antibody orcombinations thereof.

The anti-PDL-1 antibody may be selected from the group consisting ofB7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody,MEDI-4736 antibody, MSB0010718C antibody or combinations thereof.

The anti-CTLA-4 antibody may be selected from the group consisting ofipilimumab or tremelimumab or combinations thereof.

The therapeutic tumor associated antigen specific antibody may be murinemonoclonal antibody (xenotypic), a chimeric monoclonal antibody, ahumanized monoclonal antibody or a fully human monoclonal antibody.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have a constant region that may be of human origin.

The therapeutic monoclonal antibody specific for a tumor associatedantigen may have variable regions that are of human origin, non-humanorigin or any combination thereof.

The cancer may be selected from pancreatic cancer, breast cancer,colorectal cancer, ovarian cancer, renal cancer, prostate cancer,bladder cancer, gastrointestinal cancer, lung cancer and multiplemyeloma.

The tumor associated antigen may be CA125, folate binding protein (FBP),HER2/neu, MUC1 or PSA.

The following terms are defined below.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. Such term inrelation to pharmaceutical composition or other compositions in general,is intended to encompass a product comprising the active ingredient(s)and the inert ingredient(s) that make up the carrier, as well as anyproduct which results, directly or indirectly, from combination,complexation or aggregation of any two or more of the ingredients, orfrom dissociation of one or more of the ingredients, or from other typesof reactions or interactions of one or more of the ingredients.Accordingly, the pharmaceutical compositions or other compositions ingeneral of the present invention encompass any composition made byadmixing a compound of the present invention and a pharmaceuticallyacceptable carrier. By “pharmaceutically acceptable” or “acceptable” itis meant the carrier, diluent or excipient must be compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

In some embodiments, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theantibody or fragment thereof, preferably in purified form, together witha suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

The terms “inhibit”, “inhibition” or “inhibiting” as used herein in thecontext of the invention means to slow, hinder, restrain reduce orprevent. For example, “inhibiting growth” of a tumor cell as that termis used herein means to slow, hinder, restrain, reduce or prevent thetumor cell from growing.

The term “administering” as used herein refers to any action thatresults in exposing or contacting a composition containing a therapeuticmonoclonal antibody specific for a tumor associated antigen incombination with at least one immunostimulatory compound, and at leastone immune homeostatic checkpoint inhibitor, according to the inventionwith a pre-determined cell, cells, or tissue, typically mammalian. Asused herein, administering may be conducted in vivo, in vitro, or exvivo. For example, a composition may be administered by injection orthrough an endoscope. Administering also includes the direct applicationto cells of a composition according to the present invention. Forexample, during the course of surgery, tumor cells may be exposed. Inaccordance with an embodiment of the invention, these exposed cells (ortumors) may be exposed directly to a composition of the presentinvention, e.g., by washing or irrigating the surgical site and/or thecells, or by direct intra-tumoral injection of the therapeuticmonoclonal antibody specific for a tumor associated antigen incombination with at least one immunostimulatory compound, and at leastone immune homeostatic checkpoint inhibitor individually or in amixture.

The term “epitope” is intended to mean the portion of an antigen capableof being recognized by and bound by an antibody at one or more of theantibody's binding regions. Epitopes generally comprise chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and have specific three dimensional structure characteristics aswell as specific charge characteristics. In one embodiment, an epitopeof an antigen is a repetitive epitope. In one embodiment an epitope ofan antigen is a non-repetitive epitope.

The term “subject” as used herein, is a human patient or other animalsuch as another mammal with functional mast cells, basophils,neutrophils, eosinophils, monocytes, macrophages, dendritic cells, andLangerhans cells. In humans, the appropriate cells express the highaffinity receptor for IgG for the administered IgG antibody of theinvention, as well as IgE (FcεRI) for the administered IgE antibody ofthe invention.

As used herein, a reduction in growth kinetics, or complete eliminationof, a cancer tumor or a metastasized cell or tumor as used herein isdefined to mean that which is as understood in the art. For example, areduction in growth kinetics means a reduction in the exponentialgrowth, specific growth rate, or doubling time of a primary solid tumor,metastasized cell, or metastasized tumor relative to the exponentialgrowth, specific growth rate, or doubling time normally observed in vivoor in vitro for a given tumor type. Complete elimination of a tumor isthe absence of tumor presence, either by symptoms, physical exam, orradiographic imaging, in the presence of the therapeutic monoclonalantibody specific for a tumor associated antigen in combination with atleast one immunostimulatory compound, and at least one immunehomeostatic checkpoint inhibitor, where a tumor was previously seen tobe present by these detection methodologies.

The term “tumor-associated antigen” (TAA) as used herein can be any typeof cancer antigen that may be associated with a tumor as is known in theart and includes antigens found on the cell surface, including tumorcells, as well as soluble cancer antigens. Several cell surface antigenson tumors and normal cells have soluble counterparts. Such antigensinclude, but are not limited to those found on cancer-associatedfibroblasts (CAFs), tumor endothelial cells (TEC) and tumor-associatedmacrophages (TAM). Examples of cancer-associated fibroblasts (CAFs)target antigens include but are not limited to: carbonic anhydrase IX(CAIX); fibroblast activation protein alpha (FAPα); and matrixmetalloproteinases (MMPs) including MMP-2 and MMP-9. Examples of Tumorendothelial cell (TECs) target antigens include, but are not limited tovascular endothelial growth factor (VEGF) including VEGFR-1, 2, and 3;CD-105 (endoglin), tumor endothelia markers (TEMs) including TEM1 andTEM8; MMP-2; Survivin; and prostate-specific membrane antigen (PMSA).Examples of tumor associated macrophage antigens include, but are notlimited to: CD105; MMP-9; VEGFR-1, 2, 3 and TEM8. According to someembodiments, the tumor associated antigen may be CA125, folate bindingprotein (FBP), HER2/neu, MUC1 or PSA.

Before describing the present invention in detail, a number of termswill be defined. As used herein, the singular forms “a”, “an”, and “the”include plural referents unless the context clearly dictates otherwise.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that can or cannot be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that can be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation can vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present disclosure will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1 illustrates the Mucin polypeptide backbone with a Chou-Fasmanpredicted structural characteristics of the tandem repeat domain.

FIG. 2 illustrates the experimental design for the use of thecombination of an antigen specific IgG with TLR3 agonists and checkpointinhibitors.

FIG. 3 illustrates the time to tumor appearance post 1^(st) challengewith MUC1-Panc02 tumors cells for a third group of animals. Treatmentwith A) AR20.5 alone, versus control; B) AR20.5 and anti-PDL-1, versuscontrol; C) AR20.5, anti-PDL-1 and PolyICLC, versus control; D) AR20.5and PolyICLC, versus control; E) anti-PDL-1 alone, versus control; F)anti-PDL-1 and PolyICLC, versus control. Full line represents control,while dashed line represents treatment conditions.

FIG. 4 illustrates the tumor growth curves for different treatmentgroups post 1^(st) challenge with MUC1-Panc02. The tumor volume isplotted over time, over a period of 55 days.

FIG. 5 illustrates the tumor growth curves for surviving mice fromdifferent treatment groups following re-challenge with MUC1-Panc02. Thetumor volume from the MUC-1-Panco-2 challenged flank is plotted over aperiod of 46 days.

FIG. 6 illustrates the tumor growth curves for surviving mice fromdifferent treatment following re-challenge with Neo-Panc02 in theopposite flank. The tumor volume from the neo-panc02 over a period of 46days.

FIG. 7 illustrates images of MUC1 and neo-control tumors from micere-challenged having resisted primary tumor challenge with combinationtreatment. A control mouse shows the comparability of MUC1-panco2 andneo-panco-2 tumors growing in a non-immunized animal. In the treatedmice the immune resistance tumors that do not express MUC1 (Neo Tumor)grow faster than tumor cells that express MUC1 (MUC1 tumor) confirmingthe antigen specificity of the prior immunization with AR20.5 andPolyICLC or AR20.5, PolyICLC and anti-PDL-1.

FIG. 8 illustrates the adoptive transfer of splenocytes from a mousethat received AR20.5, antiPDL-1, and PolyICLC combinatorial treatmentand rejected primary tumor challenge into two MUC-1 transgenic mice. Themice were challenged with MUC1-Panc02 tumors and followed for appearanceof tumor. An untreated mouse served as the control for this challenge.The experiment confirms that that the splenocytes convey tumorresistance.

FIG. 9 illustrates the experimental design for the use of thecombination of an antigen specific IgE with TLR3 agonists and checkpointinhibitors.

FIG. 10 illustrates the time to tumor appearance post 1^(st) challengewith MUC1-Panc02 tumors cells. Treatment with A) anti-MUC1 IgE alone,versus control; B) anti-MUC1 IgE and anti-PDL-1, versus control; C)anti-MUC1 IgE, anti-PDL-1 and PolyICLC versus control; D) anti-MUC1 IgE,and PolyICLC, versus control; E) anti-PDL-1 alone, versus control; F)PolyICLC alone, versus control; and G) anti-PDL-1 and PolyICLC, versuscontrol. Full line represents control, while dashed line representstreatment condition.

FIG. 11 illustrates the tumor growth curves for different treatmentgroups post 1^(st) challenge with MUC1-Panc02 of tumors over 46 days.

FIG. 12 illustrates the tumor growth curves for MUC1 tumors postre-challenge with MUC1-Panc02 in one flank of surviving mice. A) percenttumor free for anti-MUC1 IgE, anti-PDL-1 and PolyICLC versus control;and B) tumor volume of anti-MUC1 IgE, anti-PDL-1 and PolyICLC versuscontrol, over a 57 day period.

FIG. 13 illustrates the tumor growth curves for Neo tumors postre-challenge with MUC1-Panc02 in the opposite flank of surviving mice.A) percent tumor free for anti-MUC1 IgE, anti-PDL-1 and PolyICLC versuscontrol; and B) tumor volume of anti-MUC1 IgE, anti-PDL-1 and PolyICLCversus control, over a 57 day period.

FIG. 14 illustrates images of MUC1 and neo-control tumors from the2^(nd) challenge of combination treatment immunized mice and unimmunizedcontrol mice, which confirms the antigen specificity and shows thattumors that do not express MUC1 (Neo Tumor) grow faster than tumor cellsthat express MUC1 (MUC1 tumor) in mice previously immunized withanti-MUC1 IgE and PolyICLC and anti-PDL-1who had resisted primary tumorchallenge.

DETAILED DESCRIPTION

In embodiments there are disclosed a method for treating cancer in apatient comprising administering to the patient a monoclonal antibodyspecific for a tumor associated antigen in combination with at least oneimmunostimulatory compound, and at least one immune homeostaticcheckpoint inhibitor.

The inventors have unexpectedly discovered that monoclonal antibodyspecific for a tumor associated antigen in combination with at least oneimmunostimulatory compound, and at least one immune homeostaticcheckpoint inhibitor can inhibit tumor growth. Without being bound bytheory, the combination of monoclonal antibodies specific for a tumorassociated antigen with the immunostimulatory compound, and the immunehomeostatic checkpoint inhibitor in accordance with the invention appearto be protecting subjects against growth of tumors. The invention isunique and unexpected in that it provides for a synergistic effectbetween these three immune modulators to greatly reduce and evencompletely inhibit tumor growth. The end result is that a tumor willgrow slowly or even be eliminated. This is in stark contrast to the useof these individual immune effectors alone, which are less efficient atblocking tumor cell growth.

A reduction in growth kinetics, or complete elimination of, a cancertumor or a metastasized cell or tumor as used herein is defined to meanthat which is as understood in the art. For example, a reduction ingrowth kinetics means a reduction in the exponential growth, specificgrowth rate, or doubling time of a primary solid tumor, metastasizedcell, or metastasized tumor relative to the exponential growth, specificgrowth rate, or doubling time normally observed in vivo or in vitro fora given tumor type. Complete elimination of a tumor is the absence oftumor presence, either by symptoms, physical exam, or radiographicimaging, in the presence of the therapeutic monoclonal antibody specificfor a tumor associated antigen in combination with at least oneimmunostimulatory compound, and at least one immune homeostaticcheckpoint inhibitor, where a tumor was previously seen to be present bythese detection methodologies.

According to an embodiment, antigen specific antibodies can be used toenhance T cell reactivity to self-antigens, especially in patientswithout mutations in human tumor associated antigens (TAA) that areidentical with self. By binding self-antigens with low dose immunogenicantibodies, the pool of available tumor specific T cells is enhanced andcheckpoint interference can lead to amplified immunity and enhancedclinical activity of the therapy. The use of adjuvants such as TLR3 orTLR4 agonists, in addition to selective chemotherapeutic agents thathave been found to stimulate aspects of adaptive immunity, and immunehomeostatic checkpoint inhibitors can further enhance this effect.

The combined effect of the immune modulator results in the inhibition oftumor growth and/or the facilitation of tumor destruction, in whole orin part.

A “therapeutic monoclonal antibody specific for a tumor associatedantigen” as used in the invention is a monoclonal antibody that may beany suitable monoclonal antibody, such as for example an IgG, and/or anIgE (which comprises the human Fc epsilon (ε) constant region) and alsocomprises variable regions comprising at least one antigen bindingregion specific for a tumor-associated antigen (TAA) that is a cellsurface antigen or a soluble cancer antigen located in the tumormicroenvironment or otherwise in close proximity to the tumor beingtreated.

In one embodiment, the therapeutic monoclonal antibody specific for atumor associated antigen may be specific for cancer antigens located onnon-tumor cells, for example, VEGFR-2, MMPs, Survivin, TEM8 and PMSA.The cancer antigen may be an epithelial cancer antigen, (e.g., breast,gastrointestinal, lung), a prostate specific cancer antigen (PSA) orprostate specific membrane antigen (PSMA), a bladder cancer antigen, alung (e.g., small cell lung) cancer antigen, a colon cancer antigen, anovarian cancer antigen, a brain cancer antigen, a gastric cancerantigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, aliver cancer antigen, an esophageal cancer antigen, or a head and neckcancer antigen. A cancer antigen can also be a lymphoma antigen (e.g.,non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancerantigen, a leukemia antigen, a myeloma (i.e., multiple myeloma or plasmacell myeloma) antigen, an acute lymphoblastic leukemia antigen, achronic myeloid leukemia antigen, or an acute myelogenous leukemiaantigen.

Other cancer antigens include but are not limited to mucin-1 protein orpeptide (MUC-1) that is found on most human adenocarcinomas: pancreas,colon, breast, ovarian, lung, prostate, head and neck, includingmultiple myelomas and some B cell lymphomas; human epidermal growthfactor receptor-2 (HER-2/neu) antigen; epidermal growth factor receptor(EGFR) antigen associated lung cancer, head and neck cancer, coloncancer, breast cancer, prostate cancer, gastric cancer, ovarian cancer,brain cancer and bladder cancer; prostate-specific antigen (PSA) and/orprostate-specific membrane antigen (PSMA) that are prevalently expressedin androgen-independent prostate cancers; gp-100 (Glycoprotein 100)associated with melanoma carcinoembryonic (CEA) antigen; carbohydrateantigen 19.9 (CA 19.9) related to the Lewis A blood group substance andis associated with colorectal cancers; and a melanoma cancer antigensuch as MART-1.

Other antigens include mesothelin, folate binding protein (FBP),carbohydrate antigen 125 (CA-125) and melanoma associated antigens suchas NYESO 1.

In one embodiment, the cancer antigen is a released, soluble version ofa cell surface cancer antigen. In a preferred embodiment thetumor-associated target antigen is strickly a cell surface antigenlocated on the surface of a tumor cells. In one preferred embodiment thetumor associated antigen is selected from CA125, folate binding protein(FBP), HER2/neu, MUC1, and PSA.

The terms “monoclonal antibody” or “monoclonal antibodies” as usedherein refer to a preparation of antibodies of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope. The monoclonalantibodies of the present invention are preferably chimeric, humanized,or fully human in order to bind to human antibody receptors such as thehuman Fc epsilon receptors when the subject host is a human. Humanizedand fully human antibodies are also useful in reducing immunogenicitytoward the murine components of, for example, a chimeric antibody, whenthe host subject is human. Monoclonal antibodies may be prepared bystandard techniques including, but not limited to, recombinantly andsynthetically.

The term “chimeric monoclonal antibody” refers to antibodies displayinga single binding specificity, which have one or more regions derivedfrom one antibody and one or more regions derived from another antibody.In one embodiment of the invention, the constant regions are derivedfrom the human epsilon (ε) constant region (heavy chain) and human kappaor lambda (light chain) constant regions. The variable regions of achimeric IgE monoclonal antibody of the invention are typically ofnon-human origin such as from rodents, for example, mouse (murine),rabbit, rat or hamster.

As used herein, “humanized” monoclonal antibodies comprise constantregions that are derived from human epsilon constant region (heavychain) and human kappa or lambda (light chain) constant regions. Thevariable regions of the antibodies preferably comprise a framework ofhuman origin and antigen binding regions (CDRs) of non-human origin.

Fully human or human-like antibodies may be produced through vaccinationof genetically engineered animals such as mouse lines produced at Amgen)and Bristol-Myers Squibb which contain the human immunoglobulin geneticrepertoire and produce fully human antibodies in response tovaccination. Further, the use of phage display libraries incorporatingthe coding regions of human variable regions which can be identified andselected in an antigen-screening assay to produce a human immunoglobulinvariable region binding to a target antigen.

The term “antigen binding region” refers to that portion of an antibodyas used in the invention which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody region includes the “framework”amino acid residues necessary to maintain the proper confirmation of theantigen binding residues.

An “antigen” is a molecule or portion of a molecule capable of beingbound by an antibody, which is additionally capable of inducing ananimal to produce antibody capable of binding to an epitope of thatantigen. An antigen can have one or more epitopes that are the same ordifferent. In a preferred embodiment, the antibodies of the inventionare specific for a single epitope. In one embodiment, the antigen is acapable of being bound by an antibody as used in the invention to forman immune complex that in combination with at least oneimmunostimulatory compound, and at least one immune homeostaticcheckpoint inhibitor, is capable of inhibiting cancer tumor growth. Inone embodiment, the antigen, on its own, may not be capable ofstimulating an immune response for any number of reasons, for example,the antigen is a “self” antigen, not normally recognized by the immunesystem as requiring response or the immune system has otherwise becometolerant to the antigen and does not mount an immune response. Inanother embodiment, the antigen is MUC1.

The term “epitope” is meant to refer to that portion of an antigencapable of being recognized by and bound by an antibody at one or moreof the antibody's binding regions. Epitopes generally comprisechemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structurecharacteristics as well as specific charge characteristics. In oneembodiment, an epitope of an antigen is a repetitive epitope. In oneembodiment an epitope of an antigen is a non-repetitive epitope.

Therefore, in embodiments, the therapeutic monoclonal antibody specificfor a tumor associated antigen may be any suitable antibody. Accordingto another embodiment, the therapeutic monoclonal antibody specific fora tumor associated antigen may be any suitable IgG and/or IgE antibody.According to an embodiment, the tumor associated antigen may be CA125,folate binding protein (FBP), HER2/neu, MUC1 or PSA. According toanother embodiment, the monoclonal antibody specific for a tumorassociated antigen may be for example mAb-AR20.5, mAb-B43.13, mAb3C6.hIgE, mAb 4H5.hIgE. According to another embodiment, the therapeutictumor associated antigen specific antibody may be a chimeric monoclonalantibody, a humanized monoclonal antibody or a fully human monoclonalantibody.

Methods for raising antibodies, such as murine antibodies to an antigen,and for determining if a selected antibody binds to a unique antigenepitope are well known in the art.

Screening for the desired antibody can be accomplished by techniquesknown in the art, e.g., radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (see, e.g., Antibodies—A Laboratory Manual, Harlowand Lane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor,N.Y., 1988). These include but are not limited to the hybridomatechnique originally developed by Kohler and Milstein (1975, Nature256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159: 870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314: 452-454) by splicing the genes from a mouseantibody molecule specific for a polypeptide together with genes from ahuman antibody molecule of appropriate biological activity can be used;such antibodies are within the scope of this invention.

In one embodiment, the antibody used in the invention is an IgEmonoclonal antibody comprising a nucleic acid sequence selected from aheavy chain variable region encoded by a nucleic acid sequencecomprising SEQ ID NO: 1; a light chain variable region encoded by anucleic acid sequence comprising SEQ ID NO: 2 and any combinationthereof and wherein the heavy and light chain is grafted onto humanepsilon heavy chain and kappa light chain genes, respectively.

In one embodiment the antibody used in the invention is an IgEmonoclonal antibody comprising a nucleic acid sequence selected from aheavy chain variable region encoded by the nucleic acid of SEQ ID NO: 3;a light chain variable region encoded by the nucleic acid of SEQ ID NO:4 and any combination thereof and wherein the heavy and light chain isgrafted onto human epsilon heavy chain and kappa light chain genes,respectively.

In one embodiment, the invention provides a monoclonal antibody,3C6.hIgE, comprising variable regions of the light and heavy chain ofIgG cloned from the VU-3C6 hybridoma, and grafted onto human Ig kappalight chain and epsilon heavy chain genes, respectively. VU-3C6 targetshuman mucin 1 (hMUC1), a hypoglycosylated form of mucin overexpressed ontumors arising from glandular epithelium. In one embodiment, theinvention comprises the IgE antibody, 4H5.hIgE, which is specific to anisoform of MUC1 different from the MUC1 isoform that 3C6.hIgE isspecific to.

In one embodiment, the antibody of the invention is the monoclonalantibody 3C6.hIgE comprising a heavy chain variable region encoded by anucleic acid sequence comprising SEQ ID NO: 1; a light chain variableregion encoded by a nucleic acid sequence comprising SEQ ID NO: 2.

In one embodiment the antibody of the invention is the monoclonalantibody 4H5.hIgE. The antibody 4H5.hIgE has a heavy chain variableregion encoded by the nucleic acid of SEQ ID NO: 3 and a light chainvariable region encoded by the nucleic acid of SEQ ID NO: 4 and graftedonto human Ig kappa light chain and epsilon heavy chain genes.

In one embodiment, the therapeutic monoclonal antibody specific for atumor associated antigen is an IgG monoclonal antibody specific for anepitope of MUC1. In one embodiment, the IgG monoclonal antibody isantibody AR20.5, as disclosed in Qi, W, et al.; Hybrid Hybridomics.2001; 20(5-6):313-24. MAb AR20.5 reacts strongly with either the solubleform or the cell surface epitope of MUCI on many human cancer celllines. In one embodiment, the antibody of the invention is specific forthe epitope of MUC1 comprising amino acids STAPPAHGVTSAPDTRPAPG [SEQ IDNO: 5] of MUC1. The exact epitope lies in one of the 20 amino acidrepeats that characterize the external domain of MUC1. In oneembodiment, the antibody of the invention is capable of binding MUC1 atthe epitope defined at STAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5].

In one embodiment, the therapeutic monoclonal antibody specific for atumor associated antigen is an IgE monoclonal antibody specific for anepitope of MUC1. In one embodiment, the antibody of the invention isspecific for the epitope of MUC1 comprising amino acidsSTAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5] of MUC1. The exact epitope lies inone of the 20 amino acid repeats that characterize the external domainof MUC1. In one embodiment, the antibody of the invention is capable ofbinding MUC1 at the epitope defined at STAPPAHGVTSAPDTRPAPG [SEQ ID NO:5].

In one embodiment, therapeutic monoclonal antibodies specific for atumor associated antigen in accordance with the present invention areexpressed by a positive transfectoma which is identified byenzyme-linked immunosorbent assay (ELISA) and Western Blot. The positivetransfectoma will be cloned by limited dilution for highest productivityand selected for antibody production. As used herein a “transfectoma”includes recombinant eukaryotic host cells expressing the antibody, suchas Chinese hamster ovary (CHO) cells and NS/O myeloma cells. Suchtransfectoma methodology is well known in the art (Morrison, S. (1985)Science, 229:1202). Previously published methodology used to generatemouse/human chimeric or humanized antibodies has yielded the successfulproduction of various human chimeric antibodies or antibody fusionproteins (Helguera G, Penichet M L., Methods Mol. Med. (2005)109:347-74).

In general, chimeric mouse-human monoclonal antibodies (i.e., chimericantibodies) can be produced by recombinant DNA techniques known in theart. For example, a gene encoding the Fc constant region of a murine (orother species) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.(See Robinson et al., International Patent Publication PCT/US86/02269;Akira, et al., European Patent Application 184,187; Taniguchi, M.,European Patent Application 171, 496; Morrison et al., European PatentApplication 173, 494; Neuberger et al., International Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988 Science,240:1041-1043); Liu et al. (1987) PNAS, 84:3439-3443; Liu et al., 1987,J. Immunol., 139:3521-3526; Sun et al. (1987) PNAS, 84:214-218;Nishimura et al., 1987, Canc. Res., 47:999-1005; Wood et al. (1985)Nature, 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst.,80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences ofthe Fv variable region which are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General reviews of humanized chimeric antibodies are provided byMorrison, S. L., 1985, Science, 229:1202-1207 and by Oi et al., 1986,Bio Techniques, 4:214. Those methods include isolating, manipulating,and expressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from 7E3, ananti-GPII_(b)III_(a) antibody producing hybridoma. The recombinant DNAencoding the chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable humanized antibodies canalternatively be produced by CDR substitution (U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature, 321:552-525; Verhoeyan et al. 1988 Science,239:1534; and Beidler et al. 1988 J. Immunol., 141:4053-4060).

As used herein, an “effective amount” of a therapeutic monoclonalantibody specific for a tumor associated antigen of the invention isthat amount sufficient to recognize and bind the epitope of the TAA thatis a cell surface antigen and induce, elicit, or enhance the referencedimmune response in accordance with the invention.

According to an embodiment, the present invention includesimmunostimulatory compounds. Immunostimulatory compounds are compoundhaving the capacity to stimulate or elicit an immune response. As usedherein, the term relates to exemplary immunostimulatory compounds thatinclude toll-like receptor (TLR) agonists (e.g., TLR3, TLR4, TLR7,TLR9), N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopolysaccharides (LPS), genetically modified and/or degraded LPS,alum, glucan, colony stimulating factors (e.g., EPO, GM-CSF, G-CSF,M-CSF, pegylated G-CSF, SCF, IL-3, IL6, PIXY 321), interferons (e.g.,gamma-interferon, alpha-interferon), interleukins (e.g., IL-2, IL-7,IL-12, IL-15, IL-18), MHC Class II binding peptides, saponins (e.g.,QS21), unmethylated CpG sequences, 1-methyl tryptophan, arginaseinhibitors, cyclophosphamide, antibodies that block immunosuppressivefunctions (e.g., anti-CTLA4 antibodies, anti-TGF-beta, etc.), andmixtures of two or more thereof.

In one preferred embodiment the immunostimulatory compound is a TLR3agonist. In preferred embodiments, the TLR3 agonist for use according tothe invention is a double stranded nucleic acid selected from the groupconsisting of: polyinosinic acid and polycytidylic acid, polyadenylicacid and polyuridylic acid, polyinosinic acid analogue and polycytidylicacid, polyinosinic acid and polycytidylic acid analogue, polyinosinicacid analogue and polycytidylic acid analogue, polyadenylic acidanalogue and polyuridylic acid, polyadenylic acid and polyuridylic acidanalogue, and polyadenylic acid analogue and polyuridylic acid analogue.Specific examples of double-stranded RNA as TLR3 agonists furtherinclude Polyadenur (Ipsen) and Ampligen (Hemispherx). Polyadenur is apolyA/U RNA molecule, i.e., contains a polyA strand and a polyU strand.Ampligen is disclosed for instance in EP 281 380 or EP 113 162. Inanother preferred embodiment, the TLR3 agonist may be polyIC or PolyICLC(Hiltonol®), which is a synthetic complex of carboxymethylcellulose,polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA.Poly (I:C)LC may stimulate the release of cytotoxic cytokines and, byinducing interferon-gamma production, may increase the tumoricidalactivities of various immunohematopoietic cells.

In one embodiment the immunostimulatory compound is a TLR4 agonist.Exemplary TLR4 agonists include taxanes such as paclitaxel anddocetaxal, lipopolysaccharides (LPS); E. coli LPS; and P. gingivalisLPS.

As used herein, an “effective amount” of an immunostimulatory compoundof the invention is that amount sufficient to induce, elicit, or enhancethe referenced immune response in accordance with the invention.

According to another embodiment, the present invention includes immunehomeostatic checkpoint inhibitors. Immune homeostatic checkpointinhibitors are monoclonal antibodies (mAb) directed to immune checkpointmolecules, which are expressed on immune cells and mediate signals toattenuate excessive immune reactions. According to an embodiment, immunehomeostasis checkpoint inhibition may be performed with inhibitorymonoclonal antibodies directed at the inhibitory immune receptorsCTLA-4, PD-1, and PDL-1. According to some embodiments, such inhibitorshave emerged as successful treatment approaches for patients withadvanced melanoma. According to an embodiment, the immune homeostaticcheckpoint inhibitors may be one of an anti-CTLA-4, anti-PD-1, and/oranti-PDL-1 antibody. According to an embodiment, the anti-CTLA-4antibody may be Ipilimumab or tremelimumab or combinations thereof.According to another embodiment, the anti-PDL-1 antibody may be B7-H1antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody,MEDI-4736 antibody, MSB0010718C antibody or combinations thereof.According to another embodiment, the anti-PD-1 antibody may be Nivolumabantibody, pembrolizumab antibody, pidilizumab antibody or combinationsthereof. In addition, PD-1 may also be targeted with AMP-224, which is aPD-L2-IgG recombinant fusion protein. Additional antagonists ofinhibitory pathways in the immune response are being advanced throughclinical development. IMP321 is a soluble LAG-3 Ig fusion protein andMHC class II agonist, which is used to increase an immune response totumors. LAG3 is an immune checkpoint molecule. Lirilumab is anantagonist to the KIR receptor and BMS 986016 is an antagonist of LAG3.A third inhibitory checkpoint pathway is the TIM-3-Galectin-9 pathwaythat is also a promising target for checkpoint inhibition. RX518 targetsand activates the glucocorticoid-induced tumor necrosis factor receptor(GITR), a member of the TNF receptor superfamily that is expressed onthe surface of multiple types of immune cells, including regulatory Tcells, effector T cells, B cells, natural killer (NK) cells, andactivated dendritic cells.

As used herein, an “effective amount” of an immune homeostaticcheckpoint inhibitor of the invention is that amount sufficient toinduce, elicit, or enhance the referenced immune response in accordancewith the invention.

According to another embodiment, the method of the present inventioncomprises the steps of

-   -   a) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen;    -   b) administering a therapeutically effective amount of the        immunostimulatory compound after step a); and    -   c) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor, after step b).

In one embodiment, step b) may be performed 1 or more days after stepa). In another embodiment, step c) may be performed 1 or more days afterstep b).

According to another embodiment, the method of the present inventioncomprises the steps of

-   -   a) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen;    -   b) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor after step a); and    -   c) administering, a therapeutically effective amount of the        immunostimulatory compound after step b).

In one embodiment, step b) may be performed 1 or more days after stepa). In another embodiment, step c) may be performed 1 or more days afterstep b).

According to another embodiment, the method of the present inventioncomprises the steps of

-   -   a) administering a therapeutically effective amount of the        immunostimulatory compound    -   b) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen after step a); and    -   c) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor, after step b).

In one embodiment, step b) may be performed 1 or more days after stepa). In another embodiment, step c) may be performed 1 or more days afterstep b).

According to another embodiment, the method of the present inventioncomprises the steps of

-   -   a) administering a therapeutically effective amount of the        immunostimulatory compound;    -   b) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor after step a); and    -   c) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen, after step b).

In one embodiment, step b) may be performed 1 or more days after stepa). In another embodiment, step c) may be performed 1 or more days afterstep b).

According to another embodiment, the method of the present inventioncomprises the steps of

-   -   a) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor;    -   b) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen after step a); and    -   c) administering a therapeutically effective amount of the        immunostimulatory compound, after step b).

In one embodiment, step b) may be performed 1 or more days after stepa). In another embodiment, step c) may be performed 1 or more days afterstep b).

According to another embodiment, the method of the present inventioncomprises the steps of

-   -   a) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor;    -   b) administering a therapeutically effective amount of the        immune homeostatic checkpoint inhibitor after step a); and    -   c) administering a therapeutically effective amount of the        therapeutic monoclonal antibody specific for a tumor associated        antigen, after step b).

In one embodiment, step b) may be performed 1 or more days after stepa). In another embodiment, step c) may be performed 1 or more days afterstep b).

According to another embodiment, the present invention also encompassesthe use of a therapeutic monoclonal antibody specific for a tumorassociated antigen in combination with at least one immunostimulatorycompound, and at least one immune homeostatic checkpoint inhibitor forinhibiting cancer tumor growth in a patient in need thereof.

In embodiments, the use employs the therapeutic monoclonal antibodyspecific for a tumor associated antigen in combination with at least oneimmunostimulatory compound, and at least one immune homeostaticcheckpoint inhibitor as described above for inhibiting cancer tumorgrowth in a patient in need thereof

According to yet another embodiment, the present invention alsoencompasses composition for use for inhibiting cancer tumor growth in apatient in need thereof, the composition comprising a therapeuticmonoclonal antibody specific for a tumor associated antigen, at leastone immunostimulatory compound, and at least one immune homeostaticcheckpoint inhibitor.

Such compositions comprise a therapeutically effective amount of atherapeutic monoclonal antibody specific for a tumor associated antigen,at least one immunostimulatory compound, and at least one immunehomeostatic checkpoint inhibitor and may also include a pharmaceuticallyacceptable carrier. In one preferred embodiment, the pharmaceuticalcomposition comprises a therapeutic IgE monoclonal antibody thatspecifically binds a single epitope of MUC1.

In accordance with a method or use of the invention compositionscomprising the therapeutic monoclonal antibody specific for a tumorassociated antigen, the immunostimulatory compound, and the immunehomeostatic checkpoint inhibitor of the invention may be administered tothe patient by any immunologically suitable route. For example, they maybe introduced into the patient by an intravenous, subcutaneous,intraperitoneal, intrathecal, intravesical, intradermal, intramuscular,or intralymphatic routes, alone or as combination. The composition maybe in solution, tablet, aerosol, or multi-phase formulation forms.Liposomes, long-circulating liposomes, immunoliposomes, biodegradablemicrospheres, micelles, or the like may also be used as a carrier,vehicle, or delivery system. Furthermore, using ex vivo procedures wellknown in the art, blood or serum from the patient may be removed fromthe patient; optionally, it may be desirable to purify the antigen inthe patient's blood; the blood or serum may then be mixed with acomposition that includes a binding agent according to the invention;and the treated blood or serum is returned to the patient. The inventionshould not be limited to any particular method of introducing thebinding agent into the patient.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition of the invention which will be effectivein the treatment, inhibition and prevention of tumor growth associatedwith the antigen to which the antibody of the invention is specific canbe determined by standard clinical techniques. The presence of theantibody in the extra vascular space, can be assayed by standard skinwheal and flair responses, in response to intradermal administration ofpurified antigen (e.g. MUC1). In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For the antibodies used in the invention, the dosage administered to apatient is typically 0.001 μg/kg to 1 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between 0.01μg/kg and 0.1 mg/kg of the patient's body weight, more preferably 0.02μg/kg to 20 μg/kg of the patient's body weight. Lower dosages of theantibodies of the invention and less frequent administration may also bepossible.

For the immunostimulatory compound used in the invention, the dosageadministered to a patient may be according to the ranges orconcentrations that have been optimized by their respectivemanufacturers.

For the immune homeostatic checkpoint inhibitor used in the invention,the dosage administered to a patient may be according to the ranges orconcentrations that have been optimized by their respectivemanufacturers.

The pharmaceutical compositions of the present invention have in vitroand in vivo diagnostic and therapeutic utilities. For example, thesemolecules can be administered to cells in culture, e.g., in vitro or exvivo, or in a subject, e.g., in vivo, to treat cancer. As used herein,the term “subject” is intended to include human and non-human animals. Apreferred subject is a human patient with cancer. As used herein theterms “treat” “treating” and “treatment” of cancer includes: preventingthe appearance of tumor metastasis in a patient, inhibiting the onset ofcancer in a patient; eliminating or reducing a preexisting tumor burdenin a patient either with metastatic cancer or cancer localized to theorgan of origin; prolonging survival in a cancer patient; prolonging theremission period in a cancer patient following initial treatment withchemotherapy and/or surgery; and/or prolonging any period between cancerremission and cancer relapse in a patient.

When used for therapy for the treatment of cancer, the antibodies usedin the invention are administered to the patient in therapeuticallyeffective amounts (i.e. amounts needed to treat clinically apparenttumors, or prevent the appearance of clinically apparent tumor, eitherat the original site or a distant site, at some time point in thefuture). The antibodies used in the invention and the pharmaceuticalcompositions containing them will normally be administered parenterally,when possible, or at the target cell site, or intravenously.

According to yet another embodiment, the present invention alsoencompasses kits for use for inhibiting cancer tumor growth in a patientin need thereof. The kits may comprise a therapeutic monoclonal antibodyspecific for a tumor associated antigen, at least one immunostimulatorycompound, at least one immune homeostatic checkpoint inhibitor, andinstructions on how to use the kit.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE 1 In Vivo Tumor Challenge Studies in Mice Immunized by AntigenSpecific IgG and Combinations

In order to first establish the principle of this therapeutic approach,an antibody with suitable specificity and tumor antigen expressing tumormodel to be evaluated in an animal tolerant to that tumor antigen arerequired. A Panc02 tumor cell line transfected with the human MUC1 gene(panc02.muc1) is selected. The panc02 tumors are syngeneic to BL6 miceand the panc02.muc1 is fully syngeneic to the BL6.Tg mice transgenic forhuman MUC1. The antibody used for the demonstration experiment ismAb-AR20.5 from Quest PharmaTech, a murine monoclonal antibodypreviously demonstrated to induce immunity to its ligand MUC1 inmultiple experimental systems. AR20.5 is an IgG 1κ monoclonal antibodythat binds to the sequence DTRPAP in the Core Tandem Repeat of MUC1 (SeeFIG. 1).

Experimental Design

1) MUC1.Tg animals (i.e. immunologically tolerant to MUC1): all animalsare challenged with 1×10⁶ Panc02.MUC1 cells subcutaneously.

2) mAb-AR20.5 (100 μg) is injected IV at days 7, 17, 27 and every 10days thereafter until disease progression or 47 days.

3) 50 μg PolyICLC (Hiltonol®) is given IV 7 days post tumor challenge,then at day 12, day 17, day 22, and every 5 days until diseaseprogression or 47 days.

4) 200 μg anti-PDL-1 (clone 10F.9G2 BioXL) is given IP at days 8, 10,13, 15, 18, 20, 23, 25, and the same cycle (one day and three days postHiltonol®) until disease progression or 47 days.

See FIG. 2 for an illustration of the experimental design of the presentexample and the rechallenge for mice showing tumor resistance in example2.

The results of the experiment plotted by percent tumor free over time(FIG. 3) and measured tumor volume over time (FIG. 4). A dramatictreatment effect of the combinatorial therapy using AR20.5 and TLR3stimulation in conjunction with anti-PDL-1 is observed. These resultsshow a potent interaction between the three immune modulators [anti-MUC1AR20.5, anti-PDL-1, and PolyICLC], and demonstrate an unexpectedlypotent tumor growth inhibitory effect and anti-tumor effect.

EXAMPLE 2 Rechallenge of Tumor Resistant Animals to Confirm ImmuneMemory and Specificity

Animals from the primary challenge experiment (example 1) that showed nodisease progression at day 47 days received no further treatment andwere observed for 30 additional days (to 77 days). If there was still notumor growth, these surviving animals were re-challenged in one flankwith Panc02.MUC1 (1×10⁶⁾ and in the other flank with control Panc02cells (1×10⁶) not expressing MUC1, to determine if there is a memoryresponse to MUC1, and whether there is evidence of epitope spreading orgeneral immunity to other tumor antigens on Panc02 cells. Re-challengedanimals were observed for up to 60 additional days for evidence of tumorgrowth (137 days post primary challenge and60 days post-secondary tumorchallenge). (see again FIG. 2 for schema)

A notable proportion of these mice exhibited antigen-specific rejectionof MUC1-Panc02 cells but did not reject antigen negative neo controltumor cells from the opposite flank (FIGS. 5-6). Moreover, in examplesof mice that failed to completely reject a second round of MUC1-Panc02cell challenge, they generated significantly smaller MUC-1 tumors (6.0mm×3.8 mm×4.8 mm) that did not progress as compared to control tumor(18.9 mm×21.3 mm×22.3 mm) after 50 days of tumor cell challenge (seeFIG. 7).

EXAMPLE 3 Splenocytes Transfer from Tumor-Immune Mice

Splenocytes from a tumor-immune mice that had been immunized with theAR20.5+ PolyICLC +Anti-PDL-1 combination were harvested and cultured forfive days and then transferred into two fresh MUC1.Tg transgenic micethrough tail vein injection. Post transfer, the mice were challengedwith 2×10⁶ cells/ml MUC1-Panc02 cells monitored the growth of tumor foradditional days. A control mouse that had not received prior treatmentor primary challenge also received the same challenge in both flanks.The results are shown in FIG. 8. One of the two mice receiving thetransferred splenocytes completely resisted the tumor challenge and inthe second mouse the appearance of tumor was delayed relative to theuntreated control mouse.

The experiment confirms that tumor specific resistance resides in thesplenic compartment.

EXAMPLE 4 Use of Combinations of Antigen Specific IgE with TLR3 Agonistsand Checkpoint Inhibitors

Double human transgenic C57BL6/J mice carrying the human transgenes forboth human MUC1 and the human FccR alpha chain were inoculatedsubcutaneously with a total of 10⁶ cells from syngeneic rat pancreatictumor cell line humuc1-Panc02 transfectoma's (expressing human MUC1).Subcutaneous nodules containing tumors appeared within 3 weeks ofinjection in this model and are followed for growth until animals aresacrificed per institutional animal care requirements.

Combinatorial Treatments included: anti-MUC1 IgE (20 μg/injection) atdays 7, 17, 27 and 37, PolyICLC (50 μg/infusion) at day 8, 13, 18, 23,28, 33, 38, 43; and anti-PDL-1 (200 μg per injection) at days 9, 11, 14,16, 19, 21, 24, 24, 26, 29, 31, 34, 36, 39, 41, 44. Eight groups of micewere treated with n=8/group. The groups were: 1=control; 2=anti-PDL-1;3=PolyICLC; 4=anti-PDL-1 and PolyICLC; 5=anti-MUC1 IgE alone;6=anti-MUC1 IgE and PolyICLC; 7=anti-MUC1 IgE and anti-PDL-1; and8=anti-MUC1 IgE and both PolyICLC and anti-PDL-1.

The experimental design is presented in FIG. 9, tumor free survivalplotted in FIG. 10 and tumor growth curves by treatment group areplotted in FIG. 11.

The results show in FIG. 10 that the combination treatment withanti-MUC1 IgE, anti-PDL-1, and PolyICLC results in a much reduced tumorgrowth over the 70+ challenge period (FIG. 10C). None of the animals ofthe other treatment conditions had a similar tumor growth pattern overthe same challenge period (FIGS. 10A-B and D-G), displaying much steepertumor growth over the same period.

FIG. 11 illustrates that that the combination treatment with anti-MUC1IgE, anti-PDL-1, and PolyICLC results in much lower tumor volumes overthe 46 days challenge period. The tumor volumes in the animals of theother treatment conditions are all larger, with the closest treatmentgroup (anti-MUC1 and anti-PDL-1) having a tumor volume approximately 6.7time larger than the treatment with anti-MUC1 IgE, anti-PDL-1, andPolyICLC, at the end point of the challenge period.

EXAMPLE 5 Tumor Antigen Specificity

In order to examine the effect of therapy on T cell memory andspecificity in the treated groups, a second round of tumor challengeswas performed with 1×10⁶ cells/ml of control (neomycin expressingPanc02-Neo-Panc02) and antigen expressing Panc02 cells-MUC1-Panc02, inmice that previously rejected Pan02.MUC1 tumors. Again, see FIG. 9 for asummary of the experimental protocol. The results of these experimentsare found in FIGS. 12-14.

None of the animals rejected the MUC1-Panc02 or the antigen negativecontrol tumor cells (FIGS. 12A and 13A). However, the mice that failedto reject a second round of MUC1-Panc02 cell challenge demonstratedsignificantly smaller tumors that did not progress as compared tocontrol tumor after 57 days of tumor cell challenge (see FIGS. 12B and13B, and FIG. 14).

The results of these experiments demonstrate an important interactionbetween the three immune modulators [anti-MUC1 IgE, anti-PDL-1, andPolyICLC], and demonstrate a potent anti-tumor effect of thecombination.

While preferred embodiments have been described above and illustrated inthe accompanying drawings, it will be evident to those skilled in theart that modifications may be made without departing from thisdisclosure. Such modifications are considered as possible variantscomprised in the scope of the disclosure.

SEQUENCE LISTING SEQ ID NO: 1 <120> 3C6.hlgE heavy chain variable: <212>DNA GCCGCCACCATGTACTTGGGACTGAACTGTGTATTCATAGTTTTTCTCTTAAATGGTGTCCAGAGTGAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAAGCAAAGCTAATAATCATGCAACATACTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGTTTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGGGGGGGTACGGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGTAAGTG SEQ ID NO: 2 <120>3C6.hlgE light chain variable: <212> DNAGCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGA AACGTAAGTSEQ ID NO: 3 <120> 4H5.hlgE monoclonal antibody heavychain variable region <212> DNAGCCGCCACCATGGGATGGAGCTGTATCATGCTCTTTTTGGTAGCAACAGCAACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGGTACTGACTTCAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCATACATGCAACTCAGCAGCCTGACATCTGCGGACTCTGCGGTCTATTACTGTACAAGGGGGGGTGATTACCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGGTAAGT SEQ ID NO: 4 <120>4H5.hlgE monoclonal antibody heavy chain variable region <212> DNAGCCGCCACCATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGG AGCTGAAACGTAAGTSEQ ID NO: 5 <120> Amino Acid Sequence of MUC1 epitope <212> Amino AcidSTAPPAHGVTSAPDTRPAPG

1-107. (canceled)
 108. A method for inhibiting growth of a cancerexpressing MUC1 on cancer cell surface in a patient comprisingadministering to the patient a therapeutic monoclonal antibody specificto MUC1, a TLR3 agonist, and an immune homeostatic checkpoint inhibitor,wherein: the therapeutic monoclonal antibody specific to MUC1 is (1)monoclonal antibody AR20.5; or (2) a monoclonal IgE antibody having aheavy chain variable region encoded by SEQ ID NO: 1 and a light chainvariable region encoded by SEQ ID NO:2; and the TLR3 agonist ispolyICLC.
 109. The method of claim 108, wherein the therapeuticmonoclonal antibody specific to MUC1 is monoclonal antibody 3C6.hIgE.110. The method of claim 108, wherein the therapeutic monoclonalantibody specific to MUC1 is monoclonal antibody AR20.5 or monoclonalantibody 3C6.hIgE, or a combination thereof.
 111. The method of claim108, wherein the therapeutic monoclonal antibody specific to MUC1 is amurine monoclonal antibody or a chimeric monoclonal antibody.
 112. Themethod of claim 111, wherein the therapeutic monoclonal antibodyspecific to MUC1 has a constant region that is of human origin.
 113. Themethod of claim 108, wherein the cancer is pancreatic cancer, breastcancer, colorectal cancer, ovarian cancer, renal cancer, prostatecancer, bladder cancer, gastrointestinal cancer, lung cancer, ormultiple myeloma.
 114. The method of claim 108, comprising: (a)administering a therapeutically effective amount of the therapeuticmonoclonal antibody specific to MUC1; (b) administering atherapeutically effective amount of the TLR3 agonist after step (a); and(c) administering a therapeutically effective amount of the anti-PD-L1antibody, after step (b).
 115. The method of claim 114, wherein step (b)is performed 1 or more days after step (a).
 116. The method of claim114, wherein step (c) is performed 1 or more days after step (b). 117.The method of claim 108, wherein the immune homeostatic checkpointinhibitor is an anti-PD-L1 antibody.
 118. The method of claim 117,wherein the anti-PD-L1 antibody comprises any one of monoclonal antibody10F.9G2, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody,MEDI-4736 antibody, MSB0010718C antibody or combinations thereof.